RESUMO
KRAS is one of the most commonly mutated proteins in cancer, and efforts to directly inhibit its function have been continuing for decades. The most successful of these has been the development of covalent allele-specific inhibitors that trap KRAS G12C in its inactive conformation and suppress tumour growth in patients1-7. Whether inactive-state selective inhibition can be used to therapeutically target non-G12C KRAS mutants remains under investigation. Here we report the discovery and characterization of a non-covalent inhibitor that binds preferentially and with high affinity to the inactive state of KRAS while sparing NRAS and HRAS. Although limited to only a few amino acids, the evolutionary divergence in the GTPase domain of RAS isoforms was sufficient to impart orthosteric and allosteric constraints for KRAS selectivity. The inhibitor blocked nucleotide exchange to prevent the activation of wild-type KRAS and a broad range of KRAS mutants, including G12A/C/D/F/V/S, G13C/D, V14I, L19F, Q22K, D33E, Q61H, K117N and A146V/T. Inhibition of downstream signalling and proliferation was restricted to cancer cells harbouring mutant KRAS, and drug treatment suppressed KRAS mutant tumour growth in mice, without having a detrimental effect on animal weight. Our study suggests that most KRAS oncoproteins cycle between an active state and an inactive state in cancer cells and are dependent on nucleotide exchange for activation. Pan-KRAS inhibitors, such as the one described here, have broad therapeutic implications and merit clinical investigation in patients with KRAS-driven cancers.
Assuntos
Neoplasias , Proteínas Proto-Oncogênicas p21(ras) , Transdução de Sinais , Animais , Camundongos , Peso Corporal , Ativação Enzimática , Mutação , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/patologia , Nucleotídeos/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/antagonistas & inibidores , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Transdução de Sinais/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Especificidade por SubstratoRESUMO
The availability of high-resolution 3D structural information is crucial for investigating guest-host systems across a wide range of fields. In the context of drug discovery, the information is routinely used to establish and validate structure-activity relationships, grow initial hits from screening campaigns, and to guide molecular docking. For the generation of protein-ligand complex structural information, X-ray crystallography is the experimental method of choice, however, with limited information on protein flexibility. An experimentally verified structural model of the binding interface in the native solution-state would support medicinal chemists in their molecular design decisions. Here we demonstrate that protein-bound ligand 1 H NMR chemical shifts are highly sensitive and accurate probes for the immediate chemical environment of protein-ligand interfaces. By comparing the experimental ligand 1 H chemical shift values with those computed from the X-ray structure using quantum mechanics methodology, we identify significant disagreements for parts of the ligand between the two experimental techniques. We show that quantum mechanics/molecular mechanics (QM/MM) molecular dynamics (MD) ensembles can be used to refine initial X-ray co-crystal structures resulting in a better agreement with experimental 1 H ligand chemical shift values. Overall, our findings highlight the usefulness of ligand 1 H NMR chemical shift information in combination with a QM/MM MD workflow for generating protein-ligand ensembles that accurately reproduce solution structural data.
Assuntos
Imageamento por Ressonância Magnética , Proteínas , Simulação de Acoplamento Molecular , Ligantes , Espectroscopia de Ressonância Magnética/métodos , Proteínas/químicaRESUMO
Transcription factors are generally considered challenging, if not "undruggable", targets but they promise new therapeutic options due to their fundamental involvement in many diseases. In this study, we aim to assess the ligandability of the C-terminal Rel-homology domain of nuclear factor of activated T cells 1 (NFAT1), a TF implicated in T-cell regulation. Using a combination of experimental and computational approaches, we demonstrate that small molecule fragments can indeed bind to this protein domain. The newly identified binder is the first small molecule binder to NFAT1 validated with biophysical methods and an elucidated binding mode by X-ray crystallography. The reported eutomer/distomer pair provides a strong basis for potential exploration of higher potency binders on the path toward degrader or glue modalities.
Assuntos
Fatores de Transcrição NFATC , Sítios de Ligação , Cristalografia por Raios X , Ligantes , Fatores de Transcrição NFATC/metabolismo , Fatores de Transcrição NFATC/química , Ligação Proteica , Domínios Proteicos , Relação Estrutura-AtividadeRESUMO
HUWE1 is a universal quality-control E3 ligase that marks diverse client proteins for proteasomal degradation. Although the giant HECT enzyme is an essential component of the ubiquitin-proteasome system closely linked with severe human diseases, its molecular mechanism is little understood. Here, we present the crystal structure of Nematocida HUWE1, revealing how a single E3 enzyme has specificity for a multitude of unrelated substrates. The protein adopts a remarkable snake-like structure, where the C-terminal HECT domain heads an extended alpha-solenoid body that coils in on itself and houses various protein-protein interaction modules. Our integrative structural analysis shows that this ring structure is highly dynamic, enabling the flexible HECT domain to reach protein targets presented by the various acceptor sites. Together, our data demonstrate how HUWE1 is regulated by its unique structure, adapting a promiscuous E3 ligase to selectively target unassembled orphan proteins.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Microsporídios/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/genética , Proteínas Fúngicas , Insetos , Microsporídios/genética , Modelos Moleculares , Conformação Proteica , Domínios Proteicos , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/genéticaRESUMO
Here, we report the fragment-based discovery of BI-9321, a potent, selective and cellular active antagonist of the NSD3-PWWP1 domain. The human NSD3 protein is encoded by the WHSC1L1 gene located in the 8p11-p12 amplicon, frequently amplified in breast and squamous lung cancer. Recently, it was demonstrated that the PWWP1 domain of NSD3 is required for the viability of acute myeloid leukemia cells. To further elucidate the relevance of NSD3 in cancer biology, we developed a chemical probe, BI-9321, targeting the methyl-lysine binding site of the PWWP1 domain with sub-micromolar in vitro activity and cellular target engagement at 1 µM. As a single agent, BI-9321 downregulates Myc messenger RNA expression and reduces proliferation in MOLM-13 cells. This first-in-class chemical probe BI-9321, together with the negative control BI-9466, will greatly facilitate the elucidation of the underexplored biological function of PWWP domains.
Assuntos
Histona-Lisina N-Metiltransferase/antagonistas & inibidores , Proteínas Nucleares/antagonistas & inibidores , Sistemas CRISPR-Cas , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular , Regulação da Expressão Gênica/efeitos dos fármacos , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Domínios Proteicos , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismoRESUMO
In contrast with the very well explored concept of structure-activity relationship, similar studies are missing for the dependency between binding kinetics and compound structure of a protein ligand complex, the structure-kinetic relationship. Here, we present a structure-kinetic relationship study of the cyclin-dependent kinase 8 (CDK8)/cyclin C (CycC) complex. The scaffold moiety of the compounds is anchored in the kinase deep pocket and extended with diverse functional groups toward the hinge region and the front pocket. These variations can cause the compounds to change from fast to slow binding kinetics, resulting in an improved residence time. The flip of the DFG motif ("DMG" in CDK8) to the inactive DFG-out conformation appears to have relatively little influence on the velocity of binding. Hydrogen bonding with the kinase hinge region contributes to the residence time but has less impact than hydrophobic complementarities within the kinase front pocket.
Assuntos
Ciclina C/química , Quinase 8 Dependente de Ciclina/química , Motivos de Aminoácidos , Domínio Catalítico , Cristalografia por Raios X , Desenho de Fármacos , Humanos , Ligação de Hidrogênio , Cinética , Ligantes , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Sais/química , Temperatura , Fatores de TempoRESUMO
The WWE domain is a relatively under-researched domain found in twelve human proteins and characterized by a conserved tryptophan-tryptophan-glutamate (WWE) sequence motif. Six of these WWE domain-containing proteins also contain domains with E3 ubiquitin ligase activity. The general recognition of poly-ADP-ribosylated substrates by WWE domains suggests a potential avenue for development of Proteolysis-Targeting Chimeras (PROTACs). Here, we present novel crystal structures of the HUWE1, TRIP12, and DTX1 WWE domains in complex with PAR building blocks and their analogs, thus enabling a comprehensive analysis of the PAR binding site structural diversity. Furthermore, we introduce a versatile toolbox of biophysical and biochemical assays for the discovery and characterization of novel WWE domain binders, including fluorescence polarization-based PAR binding and displacement assays, 15N-NMR-based binding affinity assays and 19F-NMR-based competition assays. Through these assays, we have characterized the binding of monomeric iso-ADP-ribose (iso-ADPr) and its nucleotide analogs with the aforementioned WWE proteins. Finally, we have utilized the assay toolbox to screen a small molecule fragment library leading to the successful discovery of novel ligands targeting the HUWE1 WWE domain.
Assuntos
Ubiquitina-Proteína Ligases , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/química , Humanos , Ligantes , Ligação Proteica , Sítios de Ligação , Domínios Proteicos , Modelos Moleculares , Proteínas Supressoras de Tumor/metabolismo , Proteínas Supressoras de Tumor/química , Proteínas Supressoras de Tumor/genética , Cristalografia por Raios X , Descoberta de Drogas/métodosRESUMO
The bromodomain and PHD-finger containing transcription factor (BPTF) is part of the nucleosome remodeling factor (NURF) complex and has been implicated in multiple cancer types. Here, we report the discovery of a potent and selective chemical probe targeting the bromodomain of BPTF with an attractive pharmacokinetic profile enabling cellular and inâ vivo experiments in mice. Microarray-based transcriptomics in presence of the probe in two lung cancer cell lines revealed only minor effects on the transcriptome. Profiling against a panel of cancer cell lines revealed that the antiproliferative effect does not correlate with BPTF dependency score in depletion screens. Both observations and the multi-domain architecture of BPTF suggest that depleting the protein by proteolysis targeting chimeras (PROTACs) could be a promising strategy to target cancer cell proliferation. We envision that the presented chemical probe and the related negative control will enable the research community to further explore scientific hypotheses with respect to BPTF bromodomain inhibition.
Assuntos
Neoplasias Pulmonares , Fatores de Transcrição , Animais , Camundongos , Proliferação de Células , Regulação da Expressão Gênica , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Target 2035, an international federation of biomedical scientists from the public and private sectors, is leveraging 'open' principles to develop a pharmacological tool for every human protein. These tools are important reagents for scientists studying human health and disease and will facilitate the development of new medicines. It is therefore not surprising that pharmaceutical companies are joining Target 2035, contributing both knowledge and reagents to study novel proteins. Here, we present a brief progress update on Target 2035 and highlight some of industry's contributions.
RESUMO
Activating mutations in KRAS are the most frequent oncogenic alterations in cancer. The oncogenic hotspot position 12, located at the lip of the switch II pocket, offers a covalent attachment point for KRASG12C inhibitors. To date, KRASG12C inhibitors have been discovered by first covalently binding to the cysteine at position 12 and then optimizing pocket binding. We report on the discovery of the in vivo active KRASG12C inhibitor BI-0474 using a different approach, in which small molecules that bind reversibly to the switch II pocket were identified and then optimized for non-covalent binding using structure-based design. Finally, the Michael acceptor containing warhead was attached. Our approach offers not only an alternative approach to discovering KRASG12C inhibitors but also provides a starting point for the discovery of inhibitors against other oncogenic KRAS mutants.
Assuntos
Neoplasias , Proteínas Proto-Oncogênicas p21(ras) , Humanos , Proteínas Proto-Oncogênicas p21(ras)/genética , Genes ras , Mutação , Neoplasias/genética , CisteínaRESUMO
Loss of WRN, a DNA repair helicase, was identified as a strong vulnerability of microsatellite instable (MSI) cancers, making WRN a promising drug target. We show that ATP binding and hydrolysis are required for genome integrity and viability of MSI cancer cells. We report a 2.2-Å crystal structure of the WRN helicase core (517-1,093), comprising the two helicase subdomains and winged helix domain but not the HRDC domain or nuclease domains. The structure highlights unusual features. First, an atypical mode of nucleotide binding that results in unusual relative positioning of the two helicase subdomains. Second, an additional ß-hairpin in the second helicase subdomain and an unusual helical hairpin in the Zn2+ binding domain. Modelling of the WRN helicase in complex with DNA suggests roles for these features in the binding of alternative DNA structures. NMR analysis shows a weak interaction between the HRDC domain and the helicase core, indicating a possible biological role for this association. Together, this study will facilitate the structure-based development of inhibitors against WRN helicase.
Assuntos
Domínio Catalítico , Neoplasias Colorretais/enzimologia , Neoplasias Colorretais/genética , Instabilidade de Microssatélites , Helicase da Síndrome de Werner/química , Helicase da Síndrome de Werner/genética , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Proteínas de Ciclo Celular/genética , Sobrevivência Celular/genética , Cristalização , DNA/metabolismo , Dano ao DNA/genética , Inativação Gênica , Células HCT116 , Humanos , Hidrólise , Espectroscopia de Ressonância Magnética/métodos , Modelos Moleculares , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas/genética , Transfecção , Zinco/metabolismo , Quinase 1 Polo-LikeRESUMO
Despite some impressive clinical results with immune checkpoint inhibitors, the majority of patients with cancer do not respond to these agents, in part due to immunosuppressive mechanisms in the tumor microenvironment. High levels of adenosine in tumors can suppress immune cell function, and strategies to target the pathway involved in its production have emerged. CD73 is a key enzyme involved in adenosine production. This led us to identify a novel humanized antagonistic CD73 antibody, mAb19, with distinct binding properties. mAb19 potently inhibits the enzymatic activity of CD73 in vitro, resulting in an inhibition of adenosine formation and enhanced T-cell activation. We then investigated the therapeutic potential of combining CD73 antagonism with other immune modulatory and chemotherapeutic agents. Combination of mAb19 with a PD-1 inhibitor increased T-cell activation in vitro Interestingly, this effect could be further enhanced with an agonist of the adenosine receptor ADORA3. Adenosine levels were found to be elevated upon doxorubicin treatment in vivo, which could be blocked by CD73 inhibition. Combining CD73 antagonism with doxorubicin resulted in superior responses in vivo Furthermore, a retrospective analysis of rectal cancer patient samples demonstrated an upregulation of the adenosine pathway upon chemoradiation, providing further rationale for combining CD73 inhibition with chemotherapeutic agents.This study demonstrates the ability of a novel CD73 antibody to enhance T-cell function through the potent suppression of adenosine levels. In addition, the data highlight combination opportunities with standard of care therapies as well as with an ADORA3 receptor agonist to treat patients with solid tumors.
Assuntos
5'-Nucleotidase/antagonistas & inibidores , Adenosina/uso terapêutico , Terapia de Imunossupressão/métodos , Adenosina/farmacologia , Animais , Feminino , Humanos , Camundongos , Microambiente TumoralRESUMO
Aberrant WNT pathway activation, leading to nuclear accumulation of ß-catenin, is a key oncogenic driver event. Mutations in the tumor suppressor gene APC lead to impaired proteasomal degradation of ß-catenin and subsequent nuclear translocation. Restoring cellular degradation of ß-catenin represents a potential therapeutic strategy. Here, we report the fragment-based discovery of a small molecule binder to ß-catenin, including the structural elucidation of the binding mode by X-ray crystallography. The difficulty in drugging ß-catenin was confirmed as the primary screening campaigns identified only few and very weak hits. Iterative virtual and NMR screening techniques were required to discover a compound with sufficient potency to be able to obtain an X-ray co-crystal structure. The binding site is located between armadillo repeats two and three, adjacent to the BCL9 and TCF4 binding sites. Genetic studies show that it is unlikely to be useful for the development of protein-protein interaction inhibitors but structural information and established assays provide a solid basis for a prospective optimization towards ß-catenin proteolysis targeting chimeras (PROTACs) as alternative modality.
Assuntos
Bibliotecas de Moléculas Pequenas/química , beta Catenina/antagonistas & inibidores , Sítios de Ligação , Cristalografia por Raios X , Humanos , Simulação de Dinâmica Molecular , Mapas de Interação de Proteínas/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Relação Estrutura-Atividade , beta Catenina/metabolismoRESUMO
KRAS, the most common oncogenic driver in human cancers, is controlled and signals primarily through protein-protein interactions (PPIs). The interaction between KRAS and SOS1, crucial for the activation of KRAS, is a typical, challenging PPI with a large contact surface area and high affinity. Here, we report that the addition of only one atom placed between Y884SOS1 and A73KRAS is sufficient to convert SOS1 activators into SOS1 inhibitors. We also disclose the discovery of BI-3406. Combination with the upstream EGFR inhibitor afatinib shows in vivo efficacy against KRASG13D mutant colorectal tumor cells, demonstrating the utility of BI-3406 to probe SOS1 biology. These findings challenge the dogma that large molecules are required to disrupt challenging PPIs. Instead, a "foot in the door" approach, whereby single atoms or small functional groups placed between key PPI interactions, can lead to potent inhibitors even for challenging PPIs such as SOS1-KRAS.
Assuntos
Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Proteína SOS1/metabolismo , Afatinib/química , Afatinib/metabolismo , Afatinib/uso terapêutico , Regulação Alostérica/efeitos dos fármacos , Sítios de Ligação , Domínio Catalítico , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/patologia , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/metabolismo , Humanos , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Mapas de Interação de Proteínas/efeitos dos fármacos , Proteínas Proto-Oncogênicas p21(ras)/antagonistas & inibidores , Proteínas Proto-Oncogênicas p21(ras)/genética , Quinazolinas/química , Quinazolinas/metabolismo , Quinazolinas/farmacologia , Quinazolinas/uso terapêutico , Proteína SOS1/agonistas , Proteína SOS1/antagonistas & inibidores , Proteína SOS1/genéticaRESUMO
KRAS is the most frequently mutated driver of pancreatic, colorectal, and non-small cell lung cancers. Direct KRAS blockade has proved challenging, and inhibition of a key downstream effector pathway, the RAF-MEK-ERK cascade, has shown limited success because of activation of feedback networks that keep the pathway in check. We hypothesized that inhibiting SOS1, a KRAS activator and important feedback node, represents an effective approach to treat KRAS-driven cancers. We report the discovery of a highly potent, selective, and orally bioavailable small-molecule SOS1 inhibitor, BI-3406, that binds to the catalytic domain of SOS1, thereby preventing the interaction with KRAS. BI-3406 reduces formation of GTP-loaded RAS and limits cellular proliferation of a broad range of KRAS-driven cancers. Importantly, BI-3406 attenuates feedback reactivation induced by MEK inhibitors and thereby enhances sensitivity of KRAS-dependent cancers to MEK inhibition. Combined SOS1 and MEK inhibition represents a novel and effective therapeutic concept to address KRAS-driven tumors. SIGNIFICANCE: To date, there are no effective targeted pan-KRAS therapies. In-depth characterization of BI-3406 activity and identification of MEK inhibitors as effective combination partners provide an attractive therapeutic concept for the majority of KRAS-mutant cancers, including those fueled by the most prevalent mutant KRAS oncoproteins, G12D, G12V, G12C, and G13D.See related commentary by Zhao et al., p. 17.This article is highlighted in the In This Issue feature, p. 1.
Assuntos
Neoplasias Pulmonares , Proteínas Proto-Oncogênicas p21(ras) , Linhagem Celular Tumoral , Humanos , Quinases de Proteína Quinase Ativadas por Mitógeno , Mutação , Nucleotídeos , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas p21(ras)/genéticaRESUMO
Activating mutations in the three human RAS genes, KRAS, NRAS and HRAS, are among the most common oncogenic drivers in human cancers. Covalent KRASG12C inhibitors, which bind to the switch II pocket in the 'off state' of KRAS, represent the first direct KRAS drugs that entered human clinical trials. However, the remaining 85% of non-KRASG12C-driven cancers remain undrugged as do NRAS and HRAS and no drugs targeting the 'on state' have been discovered so far. The switch I/II pocket is a second pocket for which the nanomolar inhibitor BI-2852 has been discovered. Here, we elucidate inhibitor binding modes in KRAS, NRAS and HRAS on and off and discuss future strategies to drug all RAS isoforms with this one pocket.
Assuntos
Inibidores Enzimáticos/farmacologia , Isoenzimas/antagonistas & inibidores , Neoplasias/tratamento farmacológico , Proteínas ras/antagonistas & inibidores , Inibidores Enzimáticos/química , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Neoplasias/enzimologia , Proteínas ras/genética , Proteínas ras/metabolismoRESUMO
Natural killer (NK) cells play a pivotal role in controlling cancer. Multiple extracellular receptors and internal signaling nodes tightly regulate NK activation. Cyclin-dependent kinases of the mediator complex (CDK8 and CDK19) were described as a signaling intermediates in NK cells. Here, we report for the first time the development and use of CDK8/19 inhibitors to suppress phosphorylation of STAT1S727 in NK cells and to augment the production of the cytolytic molecules perforin and granzyme B (GZMB). Functionally, this resulted in enhanced NK-cell-mediated lysis of primary leukemia cells. Treatment with the CDK8/19 inhibitor BI-1347 increased the response rate and survival of mice bearing melanoma and breast cancer xenografts. In addition, CDK8/19 inhibition augmented the antitumoral activity of anti-PD-1 antibody and SMAC mimetic therapy, both agents that promote T-cell-mediated antitumor immunity. Treatment with the SMAC mimetic compound BI-8382 resulted in an increased number of NK cells infiltrating EMT6 tumors. Combination of the CDK8/19 inhibitor BI-1347, which augments the amount of degranulation enzymes, with the SMAC mimetic BI-8382 resulted in increased survival of mice carrying the EMT6 breast cancer model. The observed survival benefit was dependent on an intermittent treatment schedule of BI-1347, suggesting the importance of circumventing a hyporesponsive state of NK cells. These results suggest that CDK8/19 inhibitors can be combined with modulators of the adaptive immune system to inhibit the growth of solid tumors, independent of their activity on cancer cells, but rather through promoting NK-cell function.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Quinase 8 Dependente de Ciclina/antagonistas & inibidores , Quinases Ciclina-Dependentes/antagonistas & inibidores , Células Matadoras Naturais/imunologia , Leucemia Mieloide Aguda/tratamento farmacológico , Melanoma Experimental/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Animais , Apoptose , Neoplasias da Mama/enzimologia , Neoplasias da Mama/imunologia , Neoplasias da Mama/patologia , Proliferação de Células , Citotoxicidade Imunológica/imunologia , Feminino , Humanos , Células Matadoras Naturais/efeitos dos fármacos , Leucemia Mieloide Aguda/enzimologia , Leucemia Mieloide Aguda/imunologia , Leucemia Mieloide Aguda/patologia , Melanoma Experimental/enzimologia , Melanoma Experimental/imunologia , Melanoma Experimental/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos SCID , Fosforilação , Fator de Transcrição STAT1/metabolismo , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Infections with the human immunodeficiency virus, which inevitably lead to the development of AIDS, are still among the most serious global health problems causing more than 2.5 million deaths per year. In the pathophysiological processes of this pandemic, HIV protease has proven to be an invaluable drug target because of its essential role in the virus' replication process. By use of pyrrolidine as core structure, symmetric 3,4-bis-N-alkylsulfonamides were designed and synthesized enantioselectively from D-(-)-tartaric acid as a new class of HIV protease inhibitors. Structure-guided design using the cocrystal structure of an initial lead as starting point resulted in a second series of inhibitors with improved affinity. The binding modes of four representatives were determined by X-ray crystallography to elucidate the underlying factors accounting for the SAR. With this information for further rational design, the combination of suitable side chains resulted in a final inhibitor showing a significantly improved affinity of K(i) = 74 nM.
Assuntos
Desenho de Fármacos , Inibidores da Protease de HIV/química , Inibidores da Protease de HIV/síntese química , Pirrolidinas/química , Pirrolidinas/síntese química , Ligação Competitiva , Cristalografia por Raios X , Protease de HIV/efeitos dos fármacos , Inibidores da Protease de HIV/farmacologia , Modelos Moleculares , Estrutura Molecular , Pirrolidinas/farmacologia , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
Due to the important role that aspartic proteases play in many patho-physiological processes, they have intensively been targeted by modern drug development. However, up to now, only for two family members, renin and HIV protease, approved drugs are available. Inhibitor development, mostly guided by mimicking the natural peptide substrates, resulted in very potent inhibitors for several targets, but the pharmacokinetic properties of these compounds were often not optimal. Herein we report a novel approach for lead structure discovery of non-peptidic aspartic protease inhibitors using easily accessible achiral linear oligoamines as starting point. An initial library comprising 11 inhibitors was developed and screened against six selected aspartic proteases. Several hits could be identified, among them selective as well as rather promiscuous inhibitors. The design concept was confirmed by determination of the crystal structure of two derivatives in complex with the HIV-1 protease, and represents a promising basis for the further inhibitor development.
Assuntos
Aminas/farmacologia , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Ácido Aspártico Endopeptidases/efeitos dos fármacos , Desenho de Fármacos , Inibidores de Proteases/farmacologia , Aminas/química , Secretases da Proteína Precursora do Amiloide/metabolismo , Animais , Ácido Aspártico Endopeptidases/metabolismo , Sítios de Ligação , Cristalização , Protease de HIV/efeitos dos fármacos , Pepsina A , Inibidores de Proteases/síntese química , Proteínas de Protozoários , Renina/metabolismo , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
Potent, selective and broadly characterized small molecule modulators of protein function (chemical probes) are powerful research reagents. The pharmaceutical industry has generated many high-quality chemical probes and several of these have been made available to academia. However, probe-associated data and control compounds, such as inactive structurally related molecules and their associated data, are generally not accessible. The lack of data and guidance makes it difficult for researchers to decide which chemical tools to choose. Several pharmaceutical companies (AbbVie, Bayer, Boehringer Ingelheim, Janssen, MSD, Pfizer, and Takeda) have therefore entered into a pre-competitive collaboration to make available a large number of innovative high-quality probes, including all probe-associated data, control compounds and recommendations on use (